The action and mechanism of a prolyl endopeptidase (PEP) isolated from round scad was explored. The PEP was obtained from the skeletal muscle of blue scad via separation and purification by ammonium sulfate fractionation and a series of column chromatographies, including anion-exchange chromatography using a DEAE-Sephacel column, hydrophobic interaction chromatography using a Phenyl-Sepharose column, and anion-exchange chromatography using a Q-Sepharose column. Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) results showed that the molecular weight of the PEP was 82 kDa. Peptide mass fingerprinting revealed that there were 16 peptide fragments, including a total of 169 amino acid residues. The sequence homology between the obtained peptide fragment and PEP from Neolamprologus brichardi was 98.8%, suggesting the purified enzyme was indeed a PEP. The PEP had an optimal temperature of 35°C, but showed poor thermal stability. The optimal pH of the purified enzyme was 6.0, and good stability was observed in the pH range of 5.0 to 7.5. Purified PEP was allowed to react with three synthetic fish collagen peptides, then the degraded peptides were further separated by reverse phase-high performance liquid chromatography (RP-HPLC), and electrospray ionization mass spectrometry (ESI/MS) results showed that the PEP hydrolysis site was at the carboxyl terminus of prolyl residues. The results indicate that PEP in fish muscle can collaborate with matrix metalloproteinases to further degrade collagen peptides by cleaving peptide bonds at the carboxyl side of prolyl residues, thus participating in the metabolism of fish muscle collagen. PEP is an important enzyme that participates in post-mortem fish collagen degradation.